Radiopharmaceuticals for diagnosing alzheimer&#39;s disease

ABSTRACT

This invention is directed to the use of radiopharmaceuticals in diagnosing Alzheimer&#39;s disease. In particular the radiopharmaceuticals of the invention are able to pass through the blood-brain barrier and bind to the CCR1 receptor present in brain tissue of patients having Alzheimer&#39;s disease.

[0001] This application claims benefit of U.S. Provisional ApplicationSerial No. 60/246,299, filed Nov. 6, 2000, the disclosure of which ishereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to novel radiopharmaceuticalsuseful for the diagnosis of Alzheimer's disease.

BRIEF DESCRIPTION OF THE BACKGROUND ART

[0003] Alzheimer's disease is a severe neurodegenerative disorder, andcurrently about 4 million Americans suffer from this disease. As theaging population continues to grow, this number could reach 14 millionby the middle of next century unless a cure or prevention is found. Atpresent, there is no sensitive and specific premortem test for earlydiagnosis of this disease. Alzheimer's disease is currently diagnosedbased on the clinical observation of cognitive decline, coupled with thesystematic elimination of other possible causes of those symptoms. Theconfirmation of the clinical diagnosis of “probable Alzheimer's disease”can only be made by examination of the postmortem brain. The Alzheimer'sdisease brain is characterized by the appearance of two distinctabnormal proteinaceous deposits in regions of the brain responsible forlearning and memory (e.g., cerebral cortex and hippocampus). Thesedeposits are extracellular amyloid plaques, which are characteristic ofAlzheimer's disease, and intracellular neurofibillary tangles (NFTs),which can be found in other neurodegenerative disorders as well. Amyloidpeptides are typically either 40 or 42 amino acids in length (“A¹⁻⁴⁰” or“A¹⁻⁴²”, respectively) and are formed from abnormal processing of alarger membrane-associated protein of unknown function, the amyloidprecurser protein (“APP”). Oligomeric aggregates of these peptides arethought to be neurotoxic, eventually resulting in synaptic degenerationand neuronal loss. The amount of amyloid deposition roughly correlateswith the severity of symptoms at the time of death.

[0004] In the past, there have been several attempts for the design ofradiopharmaceuticals that could be used as diagnostic agents for apremortem diagnosis of Alzheimer's disease.

[0005] Bornebroek et al. showed that the amyloid-associated proteinserum amyloid P component (SAP), labeled with ¹²³I, accumulates at lowlevels in the cerebral cortex, possibly in vessel walls, of patientswith cerebral amyloidosis (Bornebroek, M., et al., Nucl. Med. Commun.(1996), Vol. 17, pp. 929-933).

[0006] Saito et al. proposed a vector-mediated delivery of ¹²⁵I-labeledA¹⁻⁴⁰ through the blood-brain barrier. It is reported that the iodinatedA¹⁻⁴⁰ binds A amyloid plaque in tissue sections (Saito, Y., et al.,Proc. Natl. Acad. Sci. USA 1995, Vol. 92, pp.10227-10231).

[0007] U.S. Pat. No. 5,231,000 discloses antibodies with specificity toA4 amyloid polypeptide found in the brain of Alzheimer's diseasepatients. However, a method to deliver these antibodies across theblood-brain barrier has not been described.

[0008] Zhen et al. described modifications of the amyloid-binding dyeknown as “Congo Red™”, and complexes of these modified molecules withtechnetium and rhenium. The complexes with radioactive ions arepurported to be potential imaging agents for Alzheimer's disease (Zhenet al., J. Med. Chem. (1999), Vol. 42, pp. 2805-2815). However, thepotential of the complexes to cross the blood-brain barrier is limited.

[0009] A group at the University of Pennsylvania in the U.S.A.(Skovronsky, M., et al., Proc. Natl. Acad. Sci. 2000, Vol. 97, pp.7609-7614) has developed a fluorescently labeled derivative of Congo Redthat is brain permeable and that non-specifically binds to amyloidmaterials (that is, peptides in-pleated sheet conformation). Thiscompound would need to be radiolabeled and then run through pre-clinicalscreens for pharmacokinetics and toxicity before clinical testing.

[0010] Klunk et al. reported experiments with a derivative of CongoRed™, Chrysamine G (CG). It is reported that CG binds synthetic-amyloidwell in vitro, and crosses the blood-brain barrier in normal mice (Klunket al., Neurobiol. Aging (1994), Vol. 15, No. 6, pp. 691-698).

[0011] Bergström et al. presented a compound labeled with iodine-123 asa potential radioligand for visualization of M₁ and M₂ muscarinicacetylcholine receptors in Alzheimer's disease (Bergström et al., Eur.J. Nucl. Med. (1999), Vol. 26, pp. 1482-1485).

[0012] Recently, it has been discovered that certain specific chemokinereceptors are upregulated in the brains of patients with Alzheimer'sdisease (Horuk, R. et al., J. Immunol. (1997), Vol. 158, pp. 2882-2890);Xia et al., J. NeuroVirol (1999), Vol. 5, pp. 32-41). In addition, ithas been shown recently that the chemokine receptor CCR1 is upregulatedin the brains of patients with advanced Alzheimer's disease and absentin normal-aged brains (Halks-Miller et al, CCR1 Immunoreactivity inAlzheimer's Disease Brains, Society for Neuroscience Meeting Abstract,#787.6, Volume 24, 1998). Antagonists to the CCR1 receptor and their useas anti-inflammatory agents are described in the PCT Published PatentApplication, WO 98/56771.

[0013] None of the above described proposals have resulted in a clinicaldevelopment of an imaging agent for the early diagnosis of Alzheimer'sdisease. Accordingly, there is still a clinical need for a diagnosticagent that could be used for a reliable and early diagnosis ofAlzheimer's disease.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to radiopharmaceuticals thatbind to the CCR1 receptor and are able to pass through the blood-brainbarrier, and are therefore useful in diagnosing Alzheimer's disease,preferably at an early stage of the disease.

[0015] Accordingly, in one aspect, the invention is directed tocompounds of formula (I): wherein:

[0016] wherein

[0017] X¹ and X² are each independently halo;

[0018] R¹ and R² are each independently hydrogen or alkyl; and

[0019] R³ is hydrogen, amino, monoalkylamino, dialkylamino,monoaralkylamino, alkylcarbonylamino, alkenylcarbonylamino,haloalkylcarbonylamino, arylcarbonylamino, alkoxyalkylcarbonylamino,alkoxycarbonylalkylcarbonylamino, glycinamido, monoalkylglycinamido,arylcarbonylglycinamido, aminocarbonylglycinamido,(aminocarbonyl)(alkyl)glycinamido, (alkoxyalkylcarbonyl)glycinamido,ureido, monoalkylureido, monoarylureido, monoaralkylureido, oralaninamido;

[0020] and wherein either one of X¹ or X² is selected from the group of¹²³I, ¹²⁵I, ¹²⁸I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁸⁰Br and ¹⁸F; or wherein one of thecarbon atoms in the compound is ¹¹C; or a pharmaceutically acceptablesalt thereof.

[0021] In another aspect, the invention is directed to compounds offormula (II):

[0022] wherein

[0023] X¹ and X² are each independently halo;

[0024] R¹ and R² are each independently hydrogen or alkyl; and

[0025] R⁴ is hydrogen; and

[0026] R⁵ comprises a chelator capable of binding a radioactive metalatom chosen from the group of ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re;

[0027] or as a complex with ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re;

[0028] or a pharmaceutically acceptable salt thereof.

[0029] In another aspect, this invention is directed to a method ofdiagnosing Alzheimer's disease in a human which comprises administeringto a human in need of such diagnosis a compound of formula (I) orformula (II), as described above and herein, and measuring theradioactivity arising from the administration of the compound to thehuman either by using a gamma camera or by positron emission tomography(PET).

[0030] In another aspect, the invention is directed to a method of usinga compound of the invention for the manufacture of a radiopharmaceuticalfor the diagnosis of Alzheimer's disease in a human.

[0031] In another aspect, the invention is directed to a method ofpreparing compounds of the invention.

[0032] Upon further study of the specification and appended claims,further objects and advantages of this invention will become apparent tothose skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

[0033] Various other features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanyingdrawings, in which like reference characters designate the same orsimilar parts throughout the several views, and wherein:

[0034]FIG. 1 shows the expression of CCR1 in Alzheimer's disease braintissue.

[0035]FIG. 2 shows CCR1 antibody specificity in Alzheimer's diseasebrain tissue.

[0036]FIG. 3 shows CCR1-A¹⁻⁴⁰ double-labeled tissue sections

[0037]FIG. 4 shows neuritic plaques double-labeled for CCR1 and A¹⁻⁴².

[0038]FIG. 5 shows diffuse A¹⁻⁴² staining in Alzheimer's disease brain.

[0039]FIG. 6 is a graph showing the relationship between CCR1 and A¹⁻⁴⁰by CDR score.

[0040]FIG. 7 is a graph showing the relationship between CCR1 and A¹⁻⁴²by CDR score.

[0041]FIG. 8 is a graph demonstrating the decrease in total brainradioactivity over time.

[0042]FIG. 9 is a graph showing the percent of injected dose in brainand plasma.

[0043]FIG. 10 are graphs showing CCR1 expression in otherneurodegenerative diseases.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Definitions

[0045] As used in the specification and appended claims, unlessspecified to the contrary, the following terms have the meaningindicated:

[0046] “Alkyl” refers to a straight or branched chain monovalent ordivalent radical consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methyl,ethyl, n-propyl, 1-methylethyl(iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), n-heptyl, and the like.

[0047] “Alkylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(a) where R_(a) is an alkyl radical as defined above, e.g.,acetylamino, ethylcarbonylamino, n-propylcarbonylamino, and the like.

[0048] “Alkenyl” refers to a straight or branched chain monovalent ordivalent radical consisting solely of carbon and hydrogen, containing atleast one double bond and having from two to eight carbon atoms, e.g.,ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and thelike.

[0049] “Alkenylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(c) where R_(c) is an alkenyl radical as defined above,e.g., ethenylcarbonylamino, prop-2-enylcarbonylamino,but-2-enylcarbonylamino, and the like.

[0050] “Alkoxy” refers to a radical of the formula —OR_(a) where R_(a)is an alkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy(iso-propoxy), n-butoxy, n-pentoxy,1,1-dimethylethoxy(t-butoxy), and the like.

[0051] “Alkoxycarbonylalkylcarbonylamino” refers to a radical of theformula —N(H)—C(O)—Ra—C(O)OR_(a) where each R_(a) is independently analkyl radical as defined above, e.g., ethoxycarbonylmethylcarbonylamino,methoxycarbonyl methylcarbonylamino,(2-ethoxycarbonylethyl)carbonylamino,(2-methoxycarbonylethyl)carbonylamino, and the like.

[0052] “(Alkoxyalkylcarbonyl)glycinamido” refers to a radical of theformula —N(H)—C(O)-CH₂—N(H)—C(O)—R_(a)—O—R_(a) where each R_(a) isindependently an alkyl radical as defined above, e.g.,(methoxyacetyl)glycinamido, (ethoxyacetyl)glycinamido, and the like.

[0053] “Amino” refers to the radical —NH₂.

[0054] “Aminocarbonylglycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(H)—C(O)—NH₂.

[0055] “(Aminocarbonyl)(alkyl)glycinamido” refers to a radical of theformula —N(H)—C(O)—CH₂—N(R_(a))—C(O)—NH₂ where R_(a) is an alkyl radicalas defined above.

[0056] “Aryl” refers to a phenyl or naphthyl radical. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl radicalsoptionally substituted by one or more substituents selected from thegroup consisting of halo, alkyl, alkoxy, haloalkyl, haloalkoxy, nitro,amino, monoalkylamino, and dialkylamino, as defined herein.

[0057] “Arylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(b) where R_(b) is an aryl radical as defined above, e.g.,(4-methoxyphenyl)carbonylamino, (4-fluorophenyl)carbonylamino,(4-chlorophenyl)carbonylamino, and the like.

[0058] “Arylcarbonylglycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(H)—C(O)—R_(b) where R_(b) is an aryl radical as definedabove, e.g., phenylcarbonylglycinamido,(4-fluoro-3-trifluoromethylphenyl)carbonylglycinamido,(4-fluorophenyl)carbonylglycinamido, and the like.

[0059] “Aralkyl” refers to a radical of the formula —R_(a)R_(b) whereR_(a) is an alkyl radical as defined above and R_(b) is an aryl radicalas defined above, e.g., benzyl, and the like.

[0060] “Alkoxyalkylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(a)—O—R_(a) where each R_(a) is an alkyl radical as definedabove, e.g., methoxymethylcarbonylamino, ethoxyethylcarbonylamino,methoxyethylcarbonylamino, and the like.

[0061] “Alaninamido” refers to a radical of the formula—N(H)—C(O)—C(CH₃)H—NH₂.

[0062] “Benzyl” refers to a radical of the formula —CH₂—R_(h) whereR_(h) is a phenyl radical optionally substituted by one or moresubstituents selected from the group consisting of halo, alkyl,haloalkyl, alkoxy, nitro, amino, monoalkylamino, and dialkylamino.

[0063] “Dialkylamino” refers to a radical of the formula —N(R_(a))R_(a)where each R_(a) is independently an alkyl radical as defined above,e.g., dimethylamino, methylethylamino, diethylamino, dipropylamino,ethylpropylamino, and the like.

[0064] “Glycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—NH₂.

[0065] “Halo” refers to bromo, chloro, iodo or fluoro.

[0066] “Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

[0067] “Haloalkylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(f) where R_(f) is an haloalkyl radical as defined above,e.g., trifluoromethylcarbonylamino, trifluoromethylcarbonylamino,2-bromoethylcarbonylamino, and the like.

[0068] “Monoalkylamino” refers to a radical of the formula —N(H)R_(a)where R_(a) is an alkyl radical as defined above, e.g., methylamino,ethylamino, propylamino, and the like.

[0069] “Monoaralkylamino” refers to a radical of the formula —N(H)R_(d)where R_(d) is an aralkyl radical as defined above, e.g., benzylamino,(3,4,5-trimethoxybenzyl)amino, (4-chlorobenzyl)amino,and the like.

[0070] “Monoalkylglycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(H)R_(a) where R_(a) is an alkyl radical as definedabove.

[0071] “Monoalkylureido” refers to a radical of the formula—N(H)—C(O)—N(H)R_(a) or a radical of the formula —N(R_(a))—C(O)—NH₂where R_(a) is an alkyl radical as defined above.

[0072] “Monoarylureido” refers to a radical of the formula—N(H)—C(O)—N(H)R_(b) or a radical of the formula —N(R_(b))—C(O)—NH₂where R_(b) is an aryl radical as defined above

[0073] “Monoaralkylureido” refers to a radical of the formula—N(H)—C(O)—N(H)R_(d) or a radical of the formula —N(R_(d))—C(O)—NH₂where R_(d) is an aralkyl radical as defined above.

[0074] “Optional” or “optionally” means that the subsequently describedevent of circumstances may or may not occur, and that the descriptionincludes instances where said event or circumstance occurs and instancesin which it does not. For example, “optionally substituted aryl” meansthat the aryl radical may or may not be substituted and that thedescription includes both substituted aryl radicals and aryl radicalshaving no substitution.

[0075] “Pharmaceutically acceptable salt” includes both acid and baseaddition salts.

[0076] “Pharmaceutically acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases, which are not biologically or otherwise undesirable, andwhich are formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as acetic acid, propionic acid, pyruvicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Particularly preferred salts of compounds of the invention are themonochloride salts and the dichloride salts.

[0077] “Pharmaceutically acceptable base addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree acids, which are not biologically or otherwise undesirable. Thesesalts are prepared from addition of an inorganic base or an organic baseto the free acid. Salts derived from inorganic bases include, but arenot limited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, zinc, aluminum salts and the like. Preferred inorganic saltsare the ammonium, sodium, potassium, calcium, and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

[0078] “Ureido” refers to a radical of the formula —N(H)—C(O)—NH₂.

[0079] It is understood from the above definitions and examples that forradicals containing a substituted alkyl group any substitution thereoncan occur on any carbon of the alkyl group.

[0080] The compounds of the invention, or their pharmaceuticallyacceptable salts, may have asymmetric carbon atoms in their structure.The compounds of the invention and their pharmaceutically acceptablesalts may therefore exist as single enantiomers, diastereoisomers,racemates, and mixtures of enantiomers and diastereomers. All suchsingle enantiomers, diastereoisomers, racemates and mixtures thereof areintended to be within the scope of this invention. Absoluteconfiguration of certain carbon atoms within the compounds, if known,are indicated by the appropriate absolute descriptor R or S.

[0081] Separate enantiomers can be prepared through the use of opticallyactive starting materials and/or intermediates or through the use ofconventional resolution techniques, e.g., enzymatic resolution or chiralHPLC.

[0082] Utility and Administration

[0083] The compounds of the invention as described herein areantagonists to the chemokine receptor known as CCR1 and have the abilityto pass the blood-brain barrier. The compounds are therefore suited asin vivo diagnostic agents for imaging of Alzheimer's disease. Thedetection of radioactivity is performed according to well-knownprocedures in the art, either by using a gamma camera or by positronemission tomography (PET).

[0084] Preferably, the free base or a pharmaceutically acceptable saltform, e.g. a monochloride or dichloride salt, of a compound of theinvention is used in a galenical formulation as diagnostic agent. Thegalenical formulation containing the compound of the inventionoptionally contains adjuvants known in the art, e.g. buffers, sodiumchloride, lactic acid, surfactants etc. A sterilization by filtration ofthe galenical formulation under sterile conditions prior to usage ispossible.

[0085] The radioactive dose should be in the range of 1 to 100 mCi,preferably 5 to 30 mCi, and most preferably 5 to 20 mCi per application.

[0086] Testing

[0087] The suitability of the compounds as imaging agents forAlzheimer's disease can be demonstrated by experimental protocols knownto those of ordinary skill in the art. For example, the upregulation ofCCR1 receptors in Alzheimer's disease brains can be demonstrated inimmunohistochemical staining experiments of autopsy brain tissuecollected from Alzheimer's disease patients as described in detail belowin the Examples. The ability of the compounds of the invention to bindto the CCR1 receptor and their ability to pass through the blood-brainbarrier, can also be assessed in known in vitro and in vivo assays asdescribed below in the Examples. In particular, Example 10 describes alarge study that was undertaken to address the degree of CCR1 expressionin different stages of Alzheimer's disease. Brain tissue from 50 autopsycases showed a correlation between degree of clinical severity(dementia) in Alzheimer's disease and CCR1 expression in dystrophicneurites. CCR1 expression in plaque-like structures within the brains ofclinically normal individuals is rare. Also, CCR1 expression is notfound in the brains of individuals with other neurodegenerative diseasesunless there is a concomitant Alzheimer's disease pathology(specifically, Aβ¹⁻⁴² in plaques).

[0088] Preferred Embodiments

[0089] Of the various aspects of the invention, certain compounds offormula (I) are preferred. In particular, compounds of formula (I)wherein X¹ is a chloro at the 4-position of the phenyl ring and X² is a¹⁸F atom at the 4-position of the phenyl ring are preferred. Especiallypreferred are such compounds for use as diagnostic agents in positronemission tomography (PET).

[0090] Even more preferred are those compounds of formula (I) wherein R³is in the 2-position of the phenyl ring and R¹ is a methyl at the2-position of the piperazinyl ring and R² is a methyl at the 5-positionof the piperazinyl ring. Equally preferred are those compounds offormula (I) wherein R³ is in the 2-position of the phenyl ring and R¹ isa methyl in the 2-position of the piperazinyl ring and R² is hydrogen.

[0091] Further preferred are these preferred compounds in their mono- ordichloride salt form.

[0092] Of the various aspects of the invention, certain compounds offormula (II) are preferred. In particular, compounds of formula (II)wherein —N(R⁴)R⁵ is in the 2-position of the phenyl ring and R¹ is inthe 2-position of the piperazinyl ring and R² is in the 5-position ofthe piperazinyl ring. Equally preferred are those compounds of formula(II) wherein —N(R⁴)R⁵ is in the 2-position of the phenyl ring and R¹ isa methyl in the 2-position of the piperazinyl ring and R² is hydrogen.

[0093] Even more preferred are those compounds of formula (II) whereinR⁵ comprises a chelator according to formula (III):

[0094] as well as their complexes with ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re. Ofthese preferred compounds, even more preferred are those compounds offormula (II) wherein R⁵ comprises a chelator according to formula (III)or (IV), and wherein the chelator is bound to the nitrogen in the—N(R⁴)R⁵ group of the non-radioactive compound of formula (II) via alinker moiety comprising an alkyl radical having one to ten carbonatoms, wherein the alkyl radical optionally contains one to ten—C(O)-groups, one to ten —C(O)N(R)-groups, one to ten —N(R)C(O)-groups,one to ten —N(R)-groups, one to ten —N(R)₂ groups, one to ten hydroxygroups, one to ten —C(O)OR-groups, one to ten oxygen atoms, one to tensulfur atoms, one to ten nitrogen atoms, one to ten halogen atoms, oneto ten aryl groups, and one to ten saturated or unsaturated heterocyclicrings wherein R is hydrogen or alkyl. For example, the linker moiety canbe an alkyl radical having 1 to 10 carbon atoms, wherein one or more CH₂groups is optionally replaced by, in each case independently, —O—, —S—,—N(R)—, —C(O)—, —C(O)N(—R)— or —N(R)C(O)-groups. This alkyl radical canalso be optionally substituted by one or more —N(R)₂ groups, hydroxygroups, —C(O)OR groups, or halogen atoms, wherein R is hydrogen oralkyl. A preferred linker moiety is —C(O)—CH₂—N(H)—.

[0095] Of the compounds of the invention, the most preferred compoundsof formula (I) are those compounds selected from the group consisting ofthe following:

[0096]1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0097]N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,technetium-99m-complex;

[0098]1-(2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea;

[0099]N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,technetium-99m-complex;

[0100]N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,technetium-99m-complex;

[0101] 2-(2-amino-4-chlorophenoxy)-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0102] 2-(2-amino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0103] 2-(2-amino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0104] 2-(2-amino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0105]2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0106]2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0107]2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0108]2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0109] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;

[0110] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;

[0111] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;

[0112] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;

[0113] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0114] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0115] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0116] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0117] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0118] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0119] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0120] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0121]1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5,-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;

[0122]1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5,-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;

[0123]1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5,-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;

[0124]1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5,-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;

[0125] N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;

[0126] N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;

[0127] N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;

[0128] N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;

[0129] N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;

[0130] N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-11-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;

[0131] N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;

[0132] N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;

[0133] (E)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;

[0134] (E)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;

[0135] (E)-N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;

[0136] (E)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl piperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;

[0137] methyl N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0138] methyl N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0139] methyl N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0140] methyl N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0141] ethyl N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0142] ethyl N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸ F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0143] ethyl N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0144] ethyl N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;

[0145] N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0146] N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0147] N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0148] N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0149] N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;

[0150] N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;

[0151] N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;

[0152] N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;

[0153] N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;

[0154] N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;

[0155] N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;

[0156] N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;

[0157] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;

[0158] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;

[0159] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;

[0160] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;

[0161] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;

[0162] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;

[0163] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;

[0164] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;

[0165]1-benzyl-3-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0166]1-benzyl-3-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0167]1-benzyl-3-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0168]1-benzyl-3-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0169] 1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;

[0170] 1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;

[0171] 1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;

[0172] 1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;

[0173]2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0174]2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0175]2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0176]2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;

[0177]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;

[0178]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;

[0179]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;

[0180]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;

[0181] 1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0182] 1-(5-chloro-2-{2-[(2S)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;

[0183]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;

[0184]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;

[0185]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;

[0186]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;

[0187] 2-bromo-N-(5-chloro-2-{[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0188] 2-bromo-N-(5-chloro-2-{[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0189] 2-bromo-N-(5-chloro-2-{[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0190] 2-bromo-N-(5-chloro-2-{[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;

[0191]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;

[0192]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;

[0193]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;

[0194]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;

[0195]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;

[0196]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;

[0197]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;

[0198]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;

[0199] (2RS)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0200] (2SR)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0201] (2RS)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0202] (2SR)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0203] (2RS)-N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0204] (2SR)-N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0205] (2RS)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0206] (2SR)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;

[0207]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;

[0208]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;

[0209]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;

[0210]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;

[0211]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;

[0212]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;

[0213]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;

[0214]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;

[0215]N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;

[0216]N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;

[0217]N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;

[0218]N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;

[0219]N-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;and

[0220]N-(5-chloro-2-{2-[(2S)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;

[0221] as well as the mono- and dichloride salts thereof.

[0222] The compounds of the present invention may be administered by anysuitable route, preferably in the form of a pharmaceutical compositionadapted to such a route, and in a dose effective to bind to CCR1receptors in the brain and thereby be detected by gamma camera or PET.Typically, the administration is parenteral, e.g., intravenously,intraperitoneally, subcutaneously, intradermally, or intramuscularly.Intraveneous administration is preferred.

[0223] Thus, for example, the invention provides compositions forparenteral administration which comprise a solution of contrast mediadissolved or suspended in an acceptable carrier, e.g., serum orphysiological sodium chloride solution.

[0224] Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyl oleate.

[0225] Other pharmaceutically acceptable carriers, non-toxic excipients,including salts, preservatives, bufers and the like, are described, forinstance, in REMMINGTON'S PHARMACEUTICAL SCIENCES, 15^(th) Ed. Easton:Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and THE NATIONALFORMULARY XIV., 14^(th) Ed. Washington: American PharmaceuticalAssociation (1975). Aqueous carriers, are preferred.

[0226] Pharmaceutical composition of this invention are produced in amanner known per se by suspending or dissolving the compounds of thisinvention—optionally combined with the additives customary in galenicpharmacy—in an aqueous medium and then optionally sterilizing thesuspension or solution. Suitable additives are, for example,physiologically acceptable buffers (such as, for instance,tromethamine), additions of complexing agents (e.g.,diethylenetriaminepentaacetic acid) or—if required—electrolytes, e.g.,sodium chloride or—if necessary—antioxidants, such as ascorbic acid, forexample.

[0227] If suspensions or solutions of the compounds of this invention inwater or physiological saline solution are desirable for enteraladministration or other purposes, they are mixed with one or several ofthe auxiliary agents (e.g., methylcellulose, lactose, mannitol) and/ortensides (e.g., lecithins, “Tween”, “Myrj”) and/or flavoring agents toimprove taste (e.g., ethereal oils), as customary in galenic pharmacy.

[0228] The compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0229] For the compounds according to the invention having radioactivehalogens, these compounds can be shipped as “hot” compounds, i.e., withthe radioactive halogen in the compound and administered in e.g., aphysiologically acceptable saline solution. In the case of the metalcomplexes, these compounds can be shipped as “cold” compounds, i.e.,without the radioactive ion, and then mixed with Tc-generator eluate orRe-generator eluate.

[0230] Preparation of the Compounds of the Invention

[0231] A. Preparation of Compounds of Formula (I)

[0232] In general, the radioactive imaging agents of formula (I ) of thepresent invention are prepared by reacting radioactive 4-halobenzylderivatives with piperazine derivatives. Preferred are ¹⁸F-labeled4-fluorobenzyl derivatives for PET-imaging. A general method for thepreparation of 4-fluoro-¹⁸F-benzyl halides is described in Iwata et al.,Applied Radiation and Isotopes (2000), Vol. 52, pp. 87-92.

[0233] The ¹⁸F-labeled 4-fluorobenzyl derivatives are prepared byreaction of a benzaldehyde compound of formula (a) with ¹⁸F⁻ ions toobtain a benzaldehyde compound of formula (b):

[0234] wherein LG is a leaving group, for example, bromo, chloro, iodo,nitro, or N(R)₃ ⁺X⁻ (where R is alkyl and X is a halo ion, such as Br⁻,Cl⁻, or I⁻; an ion of a alkanoic acid, such as an acetate ion(CH₃C(O)O⁻); or an ion of an alkylsulfonic acid or haloalkylsulfonicacid, such as triflat (CF₃SO₃ ⁻)). Preferably LG is triflat. Startingfrom the compound of formula (b), several synthetic pathways arepossible in preparing the compounds of formula (I).

[0235] In a first synthetic pathway, a compound of formula (b) isreduced with NaBH₄ to obtain a compound of formula (c):

[0236] The compound of formula (c) is then reacted with HI, P₂I₄ orPh₃PBr₂ to obtain an iodo- or bromo-substituted compound of formulae (d)or (e):

[0237] The compounds of formulae (d) and (e) can be obtained with aradiochemical yield of 50 to 60%. The radiochemical purity is greaterthan 95%. The specific activity of the compounds is 5 mCi per 1 nmol.

[0238] A compound of formulae (d) or (e) can then be reacted with apiperazine derivative (f) to obtain a compound of formula (g) (acompound of formula (I)):

[0239] Compounds of formula (f) may be prepared according to methodsknown to those of ordinary skill in the art and is described in detailin PCT Published Patent Application, WO 98/56771.

[0240] In a second synthetic pathway, a compound of formula (b) isdirectly reacted with a compound of formula (f) using a reducing agent,for example, formic acid, ammonium formiate, NaBH₄, or NaBH₃CN, toobtain a compound of formula (g) (a compound of formula (I)):

[0241] B. Preparation of Compounds of Formula (II)

[0242] For Single Photon Emission Computed Tomography (“SPECT”),^(99m)Tc-labeled compounds are preferred. Those compounds are compoundsof formula (II). A general synthetic pathway for these compounds startswith non-radioactive analogues of compounds of formula (II) that arereacted with ^(99m)Tc-binding chelators, e.g. N₂S₂-Chelators.Preparation of the non-radioactive analogs of the compounds of formula(II) is described in detail in PCT Published Patent Application, WO98/56771. The synthesis of the chelators follows standard procedures,for example, the procedures described in A. Mahmood et al., AN₂S₂-Tetradentate Chelate for Solid-Phase Synthesis: Technetium, Rheniumin Chemistry and Nuclear Medicine (1999), Vol. 5, p. 71, or in Z. P.Zhuang et al., Bioconjugate Chemistry (1999), Vol. 10, p. 159.

[0243] Preferred chelators are chelators of formulae (III) or (IV):

[0244] One of the chelators is either bound directly to the nitrogen inthe —N(R⁴)R⁵ group of the non-radioactive compound of formula (II), orvia a linker moiety comprising an alkyl radical having one to ten carbonatoms, wherein the alkyl radical optionally contains one to ten—C(O)-groups, one to ten —C(O)N(R)-groups, one to ten —N(R)C(O)-groups,one to ten —N(R)-groups, one to ten —N(R)₂ groups, one to ten hydroxygroups, one to ten —C(O)OR-groups, one to ten oxygen atoms, one to tensulfur atoms, one to ten nitrogen atoms, one to ten halogen atoms, oneto ten aryl groups, and one to ten saturated or unsaturated heterocyclicrings wherein R is hydrogen or alkyl. A preferred linker moiety is—C(O)—CH₂—N(H)—.

[0245] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following specific examples areprovided as a guide to assist in the practice of the invention, and arenot intended as a limitation on the scope of the invention.

[0246] In the foregoing and in the following examples, all temperaturesare set forth uncorrected in degrees Celsius and, all parts andpercentages are by weight, unless otherwise indicated.

[0247] The entire disclosures of all applications, patents andpublications, cited above and below, and of corresponding U.S.Provisional Application Serial No. 60/246,299, filed Nov. 6, 2000, arehereby incorporated by reference.

EXAMPLE 1 Preparation of1-(5-Chloro-2-{2-[(2R)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea

[0248]

[0249] A. DIEA (19.10 mL, 110 mmol) was added to a solution of(R)-(−)-2-methylpiperazine (10 g, 100 mmol) in 250 mL of methylenechloride. The solution was cooled to −10° C. The BOC anhydride wasdissolved in 250 mL of methylene chloride and this solution was added tothe chilled piperazine solution over 1 hour. The reaction was allowed towarm to ambient temperature over 16 hours. The reaction mixture wasfiltered to remove the solids and the filtrate washed with 500 mL ofwater, dried over magnesium sulfate, filtered and evaporated to an oil.The oil was purified by flash column chromatography to afford 11.0 g ofthe compound of formula (a).

[0250] B. The compound of formula (a) (11.0 g, 55 mmol) and DIEA (10.5mL, 60.4 mmol) were dissolved in 100 mL of methylene chloride. Theresulting solution was chilled to −10° C. Chloroacetyl chloride (4.37mL, 55 mmol) was added dropwise to the solution maintaining thetemperature at −10° C. After stirring for 1 hour the reaction mixturewas washed with 100 mL of water, dried over magnesium sulfate, filteredand evaporated to an oil. The oil was purified by flash columnchromatography to afford 14.8 g of the compound of formula (b).

[0251] C. To a solution of the compound of formula (b) (14.8 g , 53.5mmol) and the compound of formula (e) (9.98 g, 53.5 mmol) in 75 mL ofDMSO was added potassium carbonate (18.48 g, 133.7 mmol). The resultingmixture was heated to 50° C. for 3 hours. The mixture was cooled to 30°C. and poured into 700 mL of water. The water was extracted three timeswith 200 mL of ethyl acetate. The ethyl acetate extracts were combinedand washed with 200 mL of 1N KOH followed by brine. The organic layerwas the dried over magnesium sulfate, filtered and evaporated to a foam.The foam was purified by flash column chromatography to afford 19.6 g ofthe compound of formula (c).

[0252] D. The compound of formula (c) (7.68 g, 18 mmol) was dissolved in40 mL of ethyl acetate. The resulting solution was chilled in an icebath and anhydrous HCl gas was bubbled through the solution for 5minutes. The product precipitated while the mixture was allowed to sitat ambient temperature for 1 hour. The product was collected byfiltration, washed on the filter with fresh ethyl acetate, and driedunder vacuum at ambient temperature to constant weight to afford 5.9 gof1-(5-chloro-2-{2-[(2R)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,the compound of formula (d), as white solid; NMR (2 rotomers); (400 MHz,DMSO) 9.7 (m, 0.5H), 9.2 (m, 0.5H), 8.16 (s,1 H), 8.13 (s, 0.5H), 6.8(s, 2H), 4.9 (m, 2H), 4.4 (m, 5H), 3.8 (bs, 0.5H), 3.4, (bs, 0.5H), 3.2,(m, 2.5H), 3.0 (m, 2 H) 1.2-1.4 (m, 3H) ppm.

EXAMPLE 2 Preparation of the Compound of Formula (e)

[0253]

[0254] To a solution of 2-amino-4-chlorophenol (10 g, 69.7 mmol) in 100mL of anhydrous THF at ambient temperature was added trimethylsilylisocyanate (18.8 mL, 139.4 mmol) in one portion. The solution was heatedto 60° C. and remained at this temperature for 22 hours at which timewater (1.3 mL, 76.7 mmol) was added. After 30 minutes the solution wascooled to ambient temperature and concentrated to a brown oil. This oilwas dissolved in ethyl acetate, treated with activated carbon, driedover magnesium sulfate and filtered. The filtrate was concentrated to apink solid which was crystallized from 10:1, toluene/methanol to give5.4 g of the compound of formula (e) as tan powder.

EXAMPLE 3 Preparation of1-(5-Chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea

[0255] A. Hydrogen fluoride-¹⁸F was prepared in a cyclotron bybombardment of H₂O-¹⁸O with protons. The resulting hydrogen fluoride-¹⁸Fwas adsorbed on an anion-exchange cartridge. The hydrogen fluoride-¹⁸Fwas eluted with a solution of Kryptofix 222 (15 mg, 40 mol) and K₂CO₃(2.77 mg, 20 mol) in aqueous acetonitrile (1.5 mL, 66%). The radioactivefractions were evaporated to dryness in a nitrogen gas stream. Thisprocedure was repeated three times with dry acetonitrile (1 mL). Afteraddition of a solution of 4-trimethylammonium-benzaldehyde-triflate (2mg, 6.4 pmol) in dry DMF (250 μl) the resulting reaction mixture washeated for 5 minutes to 100° C. After cooling to ambient temperature asolution of1-(5-chloro-2-{2-[(2R)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,a compound of formula (d), (3 mg, 8.3 μmol) in 50 μl acetic acid and asolution of sodium-cyano-borhydride (4 mg, 63.7 μmol) in 100 μl dry DMFwere added. The reaction mixture was heated to 120° C. for 10 minutes.After addition of 5 mL of water the mixture was filtered over apolystyrol-cartridge. The adsorbed product was washed with 2 mL of waterto afford the title compound,1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1yl]-2-oxoethoxy}phenyl)urea,which was eluted with 1.5 mL acetonitrile and purified by HPLC.

[0256] B. In a similar manner, other compounds of formula (I) containinga ¹⁸F atom are prepared.

EXAMPLE 4 Preparation ofN-(5-Chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide

[0257]

[0258] A. To a solution of (R)-(−)-2-methylpiperazine (2.0 g, 20 mmol)in 20 mL CH₂Cl₂ was added DIEA (5.2 g, 40 mmol) and 4-fluorobenzylchloride (2.39 mL, 20 mmol). The resulting mixture was stirred atambient temperature for 15 hours. After the reaction was completed, thereaction mixture was washed with water (3×20 mL) and brine, then driedover Na₂SO₄, filtered and concentrated in vacuo to afford the compoundof formula (f) (2.2 g) as a white solid.

[0259] B. To a solution of the compound of formula (f) (2.2 g, 10 mmol)in 50 mL CH₂Cl₂ was added chloroacetyl chloride (0.84 mL, 10 mmol). Theresulting mixture was stirred at ambient temperature for 10 minutes andthen triethylamine (3 mL, 21 mmol) was added. After 30 minutes, themixture was washed with water (3×20mL) and brine, then dried overNa₂SO₄, filtered and concentrated in vacuo to afford an oil.Purification by flash column chromatography afforded the compound offormula (g) (2.5 g).

[0260] C. To a solution of the compound of formula (q) (2.4 g, 8.4 mmol)in 50 mL DMF was added K₂CO₃ (2.5 g, 17 mmol), Nal (0.2 g) and4-chloro-2-nitrophenol (1.3 g, 8.4 mmol). The resulting mixture washeated at 70-80° C. After 1 hour, the mixture was concentrated in vacuo,then taken up in ethyl acetate (150 mL) and washed with water (3×100 mL)and then brine. The organic layer was separated, dried over Na₂SO₄,filtered and concentrated in vacuo to afford an oil. Purification byflash column chromatography afforded the compound of formula (h) (3.1g).

[0261] D. To a solution of the compound of formula (h) (1.1 g, 2.6 mmol)in 10 mL ethanol was added a solution of tin(II) chloride dihydrate (3.0g, 13 mmol) in 5 mL ethanol. The resulting mixture was heated at 75° C.After 1 hour, the reaction was concentrated in vacuo, then taken inethyl acetate (100 mL), washed with 1N NaOH solution in water (3×100 mL)and brine. The organic layer was separated, dried over Na₂SO₄, filteredand concentrated in vacuo to afford an oil. Purification by flash columnchromatography afforded the compound of formula (i) (0.75g).

[0262] E. To a solution of the compound of formula (i) (0.7 g, 1.78mmol) in 10 mL DMF was added BOC-Gly-OSU (0.58 g, 2.13 mmol). Theresulting mixture was heated at 50-60° C. After 24 hours, the mixturewas concentrated in vacuo, then taken up in ethyl acetate (150 mL) andwashed with water (3×100 mL) and then brine. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated in vacuo toafford an oil. Purification by flash column chromatography afforded thecompound of formula 6) (0.7 g).

[0263] F. To a solution of the compound of formula (i) (0.6 g, 1.1 mmol)in 10 mL CH₂Cl₂ was added TFA (5 mL). The resulting mixture was heatedat ambient temperature. After the reaction was completed in 1 hour, thereaction was concentrated in vacuo, then taken up in ethyl acetate (100mL), washed with 1N NaOH solution in water (2×100 mL) and brine. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated in vacuo to afford an oil. Purification by flash columnchromatography affordedN-(5-chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide,the compound of formula (k), (0.45 g) as a white solid; NMR (CDCl₃) 10.2(s, 1), 8.5 (s, 1), 7.3 (m, 2), 7.0 (m, 3), 6.8 (d, 1), 4.7 (m, 2),3.4-3.6 (m, 5), 3.0 (m, 1), 2.8 (m,1), 2.0 (m,2), 1.2-1.4 (m, 3) ppm.

EXAMPLE 5N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,Technetium-99m-complex

[0264] A. To a stirred solution ofN-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(175.5 mg, 0.4 mmol) (the compound of formula (k) as prepared above inExample 4),N-(S-trityl-2-mercaptoeth-1-yl)-N-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)glycine(291.4 mg, 0.4 mmol) (which can be synthesized according to A. Mahmoodet al., A N ₂ S ₂-Tetradentate Chelate for Solid-Phase Synthesis:Technetium, Rhenium in Chemistry and Nuclear Medicine (1999), Vol. 71),and N-hydroxysuccinimide (45.5 mg, 0.4 mmol) in 5 mL dichloromethane wasadded dropwise a solution of dicyclohexylcarbodiimide (81.4 mg, 0.4mmol) in 3 mL dichloromethane. The resulting suspension was stirred overnight at ambient temperature. After filtration, the resulting solutionwas evaporated under reduced pressure. The desired product,N′-(S-trityl-2-sulfanyleth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide(340 mg, 72.8%) was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 98:2) as a white powder. Elemental Analysis:Calc. C 69.93 H 5.87 N 7.20 O 6.85 S 5.49 Found C 69.69 H 6.05 N 7.01 OS 5.32

[0265] B.N′-(S-Trityl-2-sulfanyleth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide(116.8 mg, 0.1 mmol), as prepared above, was dissolved in 5 mLtrifluoroacetic acid. After addition of triethyl silane (48 μl, 0.3mmol) the resulting suspension was stirred for 15 minutes at ambienttemperature. The filtrate was evaporated under reduced pressure and theresidue was triturated with 7 mL diethyl ether. The precipitate wasstirred for 1 hour at ambient temperature and filtered off, yielding thedesired product,N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,bis-trifluoracetic acid salt (87 mg, 95.5%), as a white solid. ElementalAnalysis: Calc. C 44.81 H 4.65 N 9.22 O 15.80 S 7.04 Found C 44.54 H4.91 N 8.99 O S 6.80

[0266] C. Disodium tartrate (1 mg) andN′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,bis-trifluoracetic acid salt (100 μg), as prepared above, were dissolvedin 500 μl sodium phosphate buffer (0.1 M, pH=8.5). After addition of37MBq ^(99m)Tc-generator eluate, 5 μl tin-(II) chloride solution wasadded and the mixture was heated for 10 min to 100° C. HPLC analysisshowed a major peak indicating that the desired product,N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,Technetium-99m-complex, was synthesized with a RCP>90%.

EXAMPLE 6 Preparation of1-(2-{2-[(2R)-4-(4-Fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodophenyl)urea

[0267]

[0268] A. To a solution of p-iodophenol (2.2 g, 10 mmol) in 18 mL aceticacid was added a solution of HNO₃ (0.7 mL, 70%) in 5 mL acetic aciddropwise over 10 minutes. The resulting mixture was stirred at ambienttemperature. After 30 minutes, the reaction mixture was diluted by 100mL ice-water. The precipitate was collected, washed with 100 mL water.Purification by flash column chromatography afforded 1.2 g of2-nitro-4-iodophenol.

[0269] B. To a solution of the compound of formula (g) (0.3 g, 1.05mmol) (as prepared herein) in 10 mL DMF was added K₂CO₃ (0.45 g, 3.2mmol), Nal (0.01 g) and 2-nitro-4-iodophenol (0.28 g, 1.05 mmol). Theresulting mixture was heated at 70-80° C. After 1 hour, the mixture wasconcentrated in vacuo, then taken up in ethyl acetate (150 mL) andwashed with water (3×100 mL)and then brine. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated in vacuo toafford an oil. Purification by flash column chromatography afforded 0.28of the compound of formula (l).

[0270] C. To a solution of the compound of formula (l) (0.28 g, 0.55mmol) in 5 mL ethanol was added a solution of Tin(II) chloride dihydrate(0.616 g, 2.73 mmol) in 5 mL ethanol. The resulting mixture was heatedat 75° C. After 1 hour, the reaction was concentrated in vacuo, thentaken up in ethyl acetate (100 mL), washed with 1N NaOH solution inwater (3×100 mL) and brine. The organic layer was separated, dried overNa₂SO₄, filtered and concentrated in vacuo to afford an oil.Purification by flash column chromatography afforded the compound offormula (m) (0.25g).

[0271] D. To a solution of the compound of formula (m) (0.25 g, 0.52mmol) in 3 mL AcOH was added water (6 mL). The resulting mixture wasstirred at ambient temperature for 10 minutes, then a solution of KOCN(0.085 g, 1.0 mmol) in 1 mL water was added dropwise. The reactionmixture was stirred at ambient temperature for 10 minutes, then heatedat 55° C. for 5 minutes. After the reaction was completed, the reactionmixture was concentrated in vacuo, then taken up in CH₂Cl₂ (50 mL),washed with 2N NaOH solution in water (2×100 mL) and brine. The organiclayer was separated, dried over Na₂SO₄, filtered and concentrated invacuo to afford an oil. Purification by flash column chromatographyafforded the compound of formula (n) (0.16 g) as a white solid; NMR(CDCl₃) 9.0 (s, 1), 8.6 (s, 1), 7.3 (m, 2), 7.0 (t, 3), 6.6 (d, 1), 4.9(s, 2), 4.7 (m, 2), 4.4 (m, 1), 3.4-3.6 (m, 3), 3.0 (m, 1), 2.6 (d, 1),2.2 (m, 1), 2.0 (m, 1), 1.2-1.4 (m, 3) ppm.

EXAMPLE 7 Preparation of1-(2-{2-[(2R)-4-(4-Fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea

[0272] A. To a solution of the compound of formula (n) (1 mg), asprepared above, and 10 μg copper-(II)-sulfate in 300 μl DMF was added 1mCi sodium iodine[¹²³I] solution. The resulting reaction mixture washeated over night to 100° C. After adding of 1 mL half-saturated aqueousNaHCO₃ solution the product was extracted with 2 mL CH₂Cl₂. The organiclayer was evaporated to dryness in a nitrogen gas stream. The desiredproduct,1-(2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea,was purified using a RP-cartridge [eluant: EtOH/water (2:1)].

[0273] B. In a similar manner as described above, other compounds offormula (I) are prepared.

EXAMPLE 8N′-(2-Mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,Technetium-99m-complex

[0274] A. To a stirred solution of 156.7 mg (0.4 mmol)5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]aniline(compound of formula (i)) (291.4 mg, 0.4 mmol),N-(S-trityl-2-mercaptoeth-1-yl)-N-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)glycine(synthesized according to A. Mahmood et al., A N ₂ S ₂-TetradentateChelate for Solid-Phase Synthesis: Technetium, Rhenium in Chemistry andNuclear Medicine (1999), Vol. 5, p. 71) and N-hydroxysuccinimide (45.5mg, 0.4 mmol) in 5 mL dichloromethane was added dropwise a solution ofdicyclohexylcarbodiimide (81.4 mg, 0.4 mmol) in 3 mL dichloromethane.The resulting suspension was stirred over night at ambient temperature.After filtration, the resulting solution was evaporated under reducedpressure. The desired product,N′-(S-trityl-2-mercaptoeth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1yl}glycinamide(352 mg, 79.2%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 98:2) as a white powder. Elemental Analysis:Calc. C 71.36 H 5.90 N 6.30 O 5.76 S 5.77 Found C 71.08 H 6.13 N 6.05 OS 5.52

[0275] B.N′-(S-trityl-2-mercaptoeth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(111.1 mg, 0.1 mmol) was dissolved in 5 mL trifluoroacetic acid. Afteraddition of 48 μl (0.3 mmol) triethyl silane the resulting suspensionwas stirred for 15 min at ambient temperature. The fitrate wasevaporated under reduced pressure and the residue was triturated with 7mL diethyl ether. The precipitate was stirred for 1 hour at ambienttemperature and filtered off, yielding the desired product,N-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide, bis-trifluoracetic acid salt(75 mg, 87.8%) as a white solid. Elemental Analysis: Calc. C 44.99 H4.60 N 8.20 O 14.98 S 7.51 Found C 44.71 H 4.89 N 8.03 O S 7.22

[0276] C. Disodium tartrate (1 mg) disodium tartrate andN′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,bis-trifluoracetic acid salt (100 μg) were dissolved in 500 μl sodiumphosphate buffer (0.1M, pH=8.5). After addition of 37.5MBq^(99m)Tc-generator eluate, 5 μl tin-(II) chloride solution was added andthe mixture was heated for 10 minutes to 100° C. HPLC-analysis showed amajor peak indicating that the desired product, N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide, technetium -99m-complex was synthesized with aRCP>91%.

EXAMPLE 9N′-(2-Mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,Technetium-99m-complex

[0277] A. To a stirred solution of5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]aniline (156.7 mg, 0.4 mmol) and triethylamine (40.5mg, 0.4 mmol) in 5 mL dichloromethane was added dropwise a solution ofα-bromoacetyl chloride (63 mg, 0.4 mmol) in 3 mL dichloromethane. Theresulting solution was stirred over night at ambient temperature. Thesolvent was evaporated under reduced pressure. The desired product,N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}-2-bromoacetamide(175 mg, 85.3%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 99:1) as a white powder. Elemental Analysis:Calc.: C 51.53 H 4.72 N 8.19 O 5.76 Found: C 51.28 H 4.99 N 7.92 O

[0278] B. A stirred solution ofN-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}-2-bromoacetamide (165 mg, 0.42 mmol) andN,N′-bis-(S-(4-methoxybenzyl)-2-mercaptoeth-1-yl)ethylenediamine (841.3mg, 2 mmol) (synthesized according to Z. P. Zhuang et al., BioconjugateChem. (1999), Vol. 10, p. 159) in 1,4-dioxane (5 mL) was heated underreflux for 24 hours. The resulting reaction mixture was evaporated underreduced pressure. The residue was dissolved in 15 mL dichloromethane andwashed with saturated aqueous sodium carbonate solution. After dryingover MgSO₄ the solvent was evaporated under reduced pressure. Thedesired product, N′-(S-(4′-methoxybenzyl)-2-mercaptoeth-1-yl)-N′-(S-(4′-methoxybenzyl)-5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(154 mg, 43%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 9:1) as a white powder. Elemental Analysis:Calc. C 61.99 H 6.50 N 8.22 O 9.38 S 7.52 Found C 61.71 H 6.58 N 8.03 OS 7.28

[0279] C.N′-(S-(4-methoxybenzyl)-2-mercaptoeth-1-yl)-N′-(S-(4′-methoxybenzyl)-5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1yl]-2-oxoethoxy]phen-1-yl}glycinamide (140 mg, 0.164 mmol) wasdissolved at 0° C. in 5 mL trifluoroacetic acid. After addition ofHg(OAc)₂ (104.5 mg, 0.328 mmol) the resulting mixture was stirred for 30minutes at 0° C. and saturated for 15 min with H₂S. After filtration thesolvent was evaporated under reduced pressure. The resulting yellow oilwas triturated with 3 mL diethyl ether. The desired product,N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,tris-trifluoracetic acid salt (126 mg (80.5%), was isolated afterfiltration as a white powder. Elemental Analysis: Calc. C 42.79 H 4.44 N7.34 O 15.09 S 6.72 Found C 42.48 H 4.73 N 7.02 O S 6.76

[0280] D. Disodium tartrate (1 mg) andN′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,tris-trifluoracetic acid salt (100 pg) were dissolved in 500 μl sodiumphosphate buffer (0.1M, pH=8.5). After addition of 37.5MBq^(99m)Tc-generator eluate, 5 μl tin-(II) chloride solution was added andthe mixture was heated for 10 minutes to 100° C. HPLC-analysis showed amajor peak indicating that the desired product, N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,Technetium-99m-complex, was synthesized with a RCP>95%.

EXAMPLE 10 Immunohistochemical Localization of CCR1 in Human Brains

[0281] Materials and Methods

[0282] A first group of tissue samples from brains that had beenobtained at autopsy within 12 hours of death was used forimmunohistochemical staining of the chemokine receptor CCR1 inAlzheimer's disease and control brains. Paraffin-embedded, 5-μm-thick,unstained sections from frontal cortex and hippocampus were used for allimmunohistochemical and histochemical stains.

[0283] Slides were de-paraffinized in xylene and hydrated tophosphate-buffered saline containing 0.005% Triton X100 (PBS-T). Forlight microscopy (using DAB as chromogen) endogenous peroxidase activitywas blocked by incubating the slides with 0.01% H₂O₂ in methanol for 30minutes. Non-specific binding was reduced by blocking in 10% normal goatserum (NGS) in PBS for 30 minutes. The primary antibodies [Rabbitanti-CCR1, (N-terminal peptide); Mouse anti-Neurofilament; mouseanti-GFAP; mouse anti-Tau (AT8); mouse anti-CD68 (clone KP1); mouseanti-amyloid (Boehringer Mannheim, #1 381 431); Rabbit anti-CCR8,(N-terminal peptide)] were diluted in PBS-T and slides were incubatedovernight at room temperature. Staining was completed using a BiogenexABC™ kit. All washes used PBS-T. After the DAB reaction was completed,slides were lightly counterstained with Gill's Hematoxylin, dehydratedand coverslipped with Permount. Before coverslipping, some slides werere-stained with antibodies against Aβ peptide using identical methodsexcept that the chromogen was True Blue™. For immunofluorescentdouble-labeling studies the slides were de-paraffinized, blocked with10% NGS, and incubated with a cocktail of both primary antibodiesovernight. The slides were washed with PBS-T and then incubated with acocktail of goat secondary antibodies, each at 1/50 dilution. To avoidconfusion from endogenous (yellow-green) tissue fluorescence, thesecondary antibodies were conjugated to either Cy3 (_(max)=565 nm;goat-anti-mouse, Amersham) or Cy5 (_(max)=700 nm; goat-anti-rabbit,Amersham). Slides were viewed on a confocal microscope with akrypton-argon laser (model 2010, Molecular Dynamics, Sunnyvale, Calif.).

[0284] Subsequently, a second group of brain tissues composed of 10cases from cognitively normal elderly and 40 cases from Alzheimer'sdisease patients who had been assessed for clinical dementia rating(CDR) were obtained and evaluated for immunohistochemical expression ofCCR1. The samples were stained for CCR1 and other markers as describedabove except that monoclonal antibodies (clone #6D5) specific for A¹⁻⁴²(a marker for diffuse, early amyloid deposits as well as for more matureneuritic plaques) were obtained from Dr. Ursula Moenning. Theseantibodies were visualized with Vector Red™ (Vector Labs). Dr Moenningalso provided monoclonal antibodies specific for Aβ¹⁻⁴⁰ (clone #13E9), amarker for plaques found in late-stage disease, that was visualized withTrue Blue™.

[0285] Results

[0286] In both sets of brains, areas with Alzheimer's disease pathologyshowed a characteristic staining pattern. CCR1 immunoreactivity wasfound in association with neuritic (i.e., senile) plaques in both thehippocampal formation and the cerebral cortex. The immunostainedstructures were round to ovoid and were not usually associated with cellbodies. They varied in size and appeared to be filled with a punctategranular material. These structures were found to form “coronas” aroundthe amyloid deposits in senile plaques, but were distinct from the Aitself (FIGS. 1-4). The top panel in FIG. 1 shows a neuritic plaque witha corona of CCR1-positive processes. Neuronal cell bodies of some CA1and CA3 neurons in Alzheimer's disease cases were sometimes stained byCCR1 antibodies. The bottom panel in FIG. 1 illustrates this finding.This staining was distinct from neurofibrillary tangles (NFT) orgranulovacuolar bodies, although it was commonly present in the somataof neurons with these degenerative changes.

[0287] Pre-incubation of antibodies with a 16-mer polypeptide from theextracellular (N-terminus) region of the CCR1 receptor proteincompletely blocked all tissue staining, as illustrated in FIG. 2. Inparticular, the top image in FIG. 2 shows a lack of staining inAlzheimer's disease brain with CCR1 antibodies that were pre-incubatedwith the polypeptide while the bottom image in FIG. 2 shows manyCCR1-positive structures in a sister section stained with un-incubatedantibodies.

[0288] Double-labeling studies showed that nearly all CCR1-positivestructures were associated with neuritic plaques containing A¹⁻⁴² asshown in FIG. 4. A¹⁻⁴² in diffuse plaques (FIG. 5) was not typicallyassociated with CCR1 nor with any significant cellular responses. Inmore advanced cases of Alzheimer's disease, many more A¹⁻⁴⁰-positiveplaques were seen. Some, but not all of these were associated with CCR1staining as shown in FIG. 3. In some cases CCR1-positive plaques werecompletely free of Aβ¹⁻⁴⁰ staining.

[0289] In cases of severe Alzheimer's disease where significant neuronalloss and gliosis were seen, reactive astrocytes in the subiculum andentorhinal cortex were also CCR1 positive. In less severe cases,CCR1-positive reactive astrocytes were uncommon.

[0290] Confocal microscopy of double-labeled sections showed that CCR1immunoreactivity co-localized with neurofilament-positive processes. Themacrophage/microglial marker, CD68, was not associated with CCR1staining, nor was the AT8 antibody against abnormally phosphorylated tauprotein. As expected from light microscopic studies, CCR1immunoreactivity did co-localize with GFAP in areas of astrogliosis.

[0291] Leukocytes present within cerebral vessels were strongly CCR1positive in both Alzheimer's disease and control brain tissues, servingas positive internal controls for staining methods. In FIG. 5 note theCCR1-positive staining of two intravascular cells (arrows). SomeCCR1-positive material is also seen at asterisk, but in general, diffuseplaques are not associated with CCR1.

[0292] Using hippocampal tissue from the second study (where patientswere grouped into known clinical categories by CDR score), aquantitative evaluation of the number of CCR1-positive plaque-likestructures in the hippocampus was undertaken. Histologic evaluations ofindividual sections were performed under blinded conditions with respectto clinical (CDR) status. The volume analyses were conducted usingunbiased computerized methods.

[0293]FIG. 3 demonstrates the histologic relationship betweenCCR1-positive dystrophic neurites (brown stain) and a neuritic plaquecontaining A¹⁻⁴⁰ (blue stain). Note in that in FIG. 3 the CCR1-positiveprocesses are distinct from the amyloid. Plaques containing A¹⁻⁴⁰ werediscrete and easily counted, as were the clusters of CCR1-positiveneurites. Slides of hippocampus were evaluated for the number ofCCR1-positive coronas, the number of A¹⁻⁴⁰ positive plaques, and thenumber of plaques containing both markers. The structures were countedby region (e.g., CA3, CA1, and subiculum) within the entire hippocampalformation, including entorhinal cortex and then totaled. The values arerelative estimates of the number of structures in the hippocampus asrepresented by 5-μm thick cross-sections of the hippocampal formation.FIG. 6 shows the relationship between CCR1 and A¹⁻⁴⁰ by CDR score.

[0294] Quantification of the amount of A¹⁻⁴² required a differenttechnique because A¹⁻⁴² was more abundant and formed “diffuse” plaquesthat could not be easily enumerated (see FIG. 5). For these slides acomputer-assisted method (C.A.S.T. stereology system) was used toestimate the area of entorhinal cortex occupied by A¹⁻⁴² This area wasexpressed as a percent of the total area of cortex on the slide. Theareas occupied by neuritic and diffuse plaques were counted separately.The computer-driven microscope stage assured random (unbiased)evaluation of the tissue sections. The volume % of entorhinal cortexoccupied by A¹⁻⁴² (both diffuse and neuritic) is compared to the numberof CCR1-positive plaques in a sister section of entorhinal cortex inFIG. 7.

[0295]FIG. 6 shows that the average number of CCR1-positive dystrophicneurites in the hippocampus increases as a function of CDR score inAlzheimer's disease. The number of positive structures in earlyAlzheimer's disease (CDR 0.5) is increased above control (CDR 0) levels.Although the differences between control and Alzheimer's disease groupsdid not become statistically significant until group CDR 2, theseexpression patterns support the conclusion that CCR1 is upregulated indystrophic neuronal processes even at very early stages of Alzheimer'sdisease. Note that the number of A¹⁻⁴⁰ plaques do not rise until late inthe disease. CCR1 expression in brain tissue may thus be considered arelatively early indicator of Alzheimer's disease.

[0296] The correlation between number of CCR1-positive plaques inentorhinal cortex and the amount of Aβ¹⁻⁴² is shown in FIG. 7. Ingeneral, CCR1 levels in entorhinal cortex rise as the disease stateincreases; however, the area sampled is much smaller than the areasampled in FIG. 6. Note, however, that A¹⁻⁴² levels rise early in thedisease.

EXAMPLE 11 Assessment of Brain Availability, Using a ¹⁴C-labeled Tracer

[0297] A ¹⁴C analogue of 1-(5-chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea was prepared and usedfor pharmacokinetic studies of brain availability. Mice were injectedi.v. through cannulated jugular veins with the analogue at 36 mg/kgcontaining 334,000 dpm per dose. Mice (in groups of four) weresacrificed at 1, 15, 30, 120, and 1440 minutes after injection by CO₂inhalation followed by intra-cardiac puncture for removal of wholeblood. The chest was then opened and mice were perfused through theheart with phosphate buffered saline for five minutes to remove residualradioactive drug from the blood compartment. The brains were thenremoved, weighed, and sampled for amount of radioactivity in twoseparate pieces of cerebral cortex. The radioactivity levels per mg oftissue in the two samples were averaged and the data were normalized tototal brain weight, as illustrated in FIG. 8. The bars represent thestandard error (n=4 mice per time point). In particular, the graph inFIG. 8 shows that the total number of disintegrations per minute (DPM)for ¹⁴C analogue in whole mouse brain decreases to negligible levels bytwo hours after injection. At the 1-minute time point, calculations showthat, on average, about 3% of the injected dose is found in whole mousebrain (average weight of 450 g).

[0298] Plasma samples prepared from the whole blood removed from themice were also analyzed for radioactive content. The amount ofradioactivity in equal volumes of brain and plasma was compared overtime as a percent of injected dose, as illustrated in FIG. 9. The % ofinjected dose (i.d.) found per mL of plasma also declines over time,reaching negligible levels after two hours. For comparison to brainlevels, the total number of DPM per brain were normalized to a gram ofbrain weight and expressed as a percentage of the i.d. per gram of braintissue. Note that mouse brains weigh on average about 450 mg so that the% of i.d. in 1 g of mouse brain shown in the graph overestimates thereal value by about two-fold. It is clear that the levels of theanalogue in the brain fall in concert with plasma levels. Although theanalogue is lipophilic, normal mouse brain does not appear to be a depotsite for the CCR1 antagonist.

EXAMPLE 12 CCR1 Expression in Other Neurodegenerative Diseases

[0299] Materials and Methods

[0300] Histologic samples of autopsy brain tissue from a total of 29cases from seven different neurodegenerative diseases (other than pureAlzheimer's disease) were obtained and studied for CCR1 content. Slideswere immunostained with antibodies against CCR1 as described in Example10 above and reviewed under blinded conditions with respect to thespecific neurodegenerative disease. Subsequently, sister sections weredouble-labeled with antibodies against CCR1 (DAB as chromogen) and A¹⁻⁴²(Vector Red™ as chromogen). The diseases examined are listed below:Parkinson's disease (PD) 6 cases Parkinsonian dementia of Guam 3 casesCongophilic angiopathy 4 cases Multi-infarct dementia (MID) 4 casesDiffuse Lewy body dementia 4 cases (DLBD) Pick's disease 4 casesProgressive supranuclear palsy (PSP) 4 cases

[0301] The stained slides were graded for CCR1 and A¹⁻⁴² content using ahistologic scale of 0 to 4, with 0 being absence of staining, 0.5indicating rare expression, and grades 1 through 4 indicating increasinglevels of expression with 4 being highly abundant.

[0302] Results

[0303] All six cases of Parkinson's disease and all three cases ofParkinsonian dementia of Guam were negative for CCR1. CCR1-positiveplaque-like structures, similar to those found in Alzheimer's disease,were observed in all 4 cases of congophilic angiopathy, in 3 of the 4cases of DLBD, in 2 out of the 4 cases of PSP, and in 1 out of the 4cases of both MID and Pick's disease.

[0304] Double-labeling studies confirmed the assumption that theseCCR1-positive plaque-like structures were associated with A^(1-42,) aswas found in pure cases of Alzheimer's disease (Example 10). FIG. 10shows the results of the pathological evaluations in graphic form forall the diseases except for Parkinson's (which was negative for CCR1 inbrain). Basically, CCR1 expression was never found in brain tissuesamples unless A ¹⁻⁴²-positive neuritic plaques were also present.

[0305] After the code was broken, it was found that the one case ofPick's disease showing CCR1 expression also carried the diagnosis ofAlzheimer's disease. The two PSP cases with CCR1 were also diagnosedwith concurrent Alzheimer's disease. Congophilic angiopathy and DLBD arediseases that are closely associated with Alzheimer's disease pathology.Therefore, it is not surprising that they would also have high levels ofCCR1 expression in association with Aβ¹⁻⁴². In elderly populations it isoften the case that Alzheimer's disease pathology will overlay otherdisease processes. This was found in one of the four MID cases. Theseresults suggests that CCR1 is a marker that is closely associated withAlzheimer's disease pathology (specifically, Aβ¹⁻⁴²-positive neuriticplaques) regardless of other concurrent pathological processes that maybe present in brain. As such, it is likely to be highly specific forAlzheimer's disease pathology and therefore may be useful as adiagnostic surrogate marker of disease progression.

[0306] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A compound of formula (I):

wherein: X¹ and x² are each independently halo; R¹ and R² are eachindependently hydrogen or alkyl; and R³ is hydrogen, amino,monoalkylamino, dialkylamino, monoaralkylamino, alkylcarbonylamino,alkenylcarbonylamino, haloalkylcarbonylamino, arylcarbonylamino,alkoxyalkylcarbonylamino, alkoxycarbonylalkylcarbonylamino, glycinamido,monoal kylglycinamido, arylcarbonylglycinamido,aminocarbonylglycinamido, (aminocarbonyl)(alkyl)glycinamido,(alkoxyalkylcarbonyl)glycinamido, ureido, monoalkylureido,monoarylureido, monoaralkylureido, or alaninamido; and wherein eitherone of X¹ or X² is selected from the group of ¹²³I, ¹²⁵I, ¹²⁸I, ¹³¹I,⁷⁵Br, ⁷⁶Br, ⁸⁰Br and ¹⁸F; or wherein one of the carbon atoms in thecompound is ¹¹C; or a pharmaceutically acceptable salt thereof.
 2. Acompound of formula (II):

wherein X¹ and X² are each independently halo; R¹ and R² are eachindependently hydrogen or alkyl; and R⁴ is hydrogen; and R⁵ comprises achelator capable of binding a radioactive metal atom chosen from thegroup of ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re; or as a complex with ^(99m)Tc, ¹⁸⁶Reand ¹⁸⁸Re; or a pharmaceutically acceptable salt thereof.
 3. A compoundaccording to claim 1, wherein said compound binds to chemokine receptorCCR1 and passes the blood-brain barrier.
 4. A compound according toclaim 2, wherein said compound binds to chemokine receptor CCR1 andpasses the blood-brain barrier.
 5. A compound according to claim 1,wherein R¹ is methyl at the 2-position of the piperazinyl radical and R²is methyl at the 5-position of the piperazinyl radical.
 6. A compoundaccording to claim 2, wherein R¹ is methyl at the 2-position of thepiperazinyl radical and R² is methyl at the 5-position of thepiperazinyl radical.
 7. A compound according to claim 1, wherein R¹ ismethyl at the 2-position of the piperazinyl radical and R² is hydrogen.8. A compound according to claim 2, wherein R¹ is methyl at the2-position of the piperazinyl radical and R² is hydrogen.
 9. A compoundof claim 1 wherein X¹ is chloro at the 4-position of the phenyl radicaland X² is a ¹⁸F atom at the 4-position of the phenyl radical.
 10. Acompound of claim 2, wherein R⁵ comprises a chelator structure offormula (III):


11. A compound of claim 2, wherein R⁵ comprises a chelator structure offormula (IV):


12. A compound of claim 10, wherein R⁵ further comprises a linker moietycomprising an alkyl radical having one to ten carbon atoms, wherein thealkyl radical optionally contains one to ten —C(O)-groups, one to ten—C(O)N(R)-groups, one to ten —N(R)C(O)-groups, one to ten —N(R)-groups,one to ten —N(R)₂ groups, one to ten hydroxy groups, one to ten—C(O)OR-groups, one to ten oxygen atoms, one to ten sulfur atoms, one toten nitrogen atoms, one to ten halogen atoms, one to ten aryl groups,and one to ten saturated or unsaturated heterocyclic rings wherein R ishydrogen or alkyl.
 13. A compound of claim 11, wherein R⁵ furthercomprises a linker moiety comprising an alkyl radical having one to tencarbon atoms, wherein the alkyl radical optionally contains one to ten—C(O)-groups, one to ten —C(O)N(R)-groups, one to ten —N(R)C(O)-groups,one to ten —N(R)-groups, one to ten —N(R)₂ groups, one to ten hydroxygroups, one to ten —C(O)OR-groups, one to ten oxygen atoms, one to tensulfur atoms, one to ten nitrogen atoms, one to ten halogen atoms, oneto ten aryl groups, and one to ten saturated or unsaturated heterocyclicrings wherein R is hydrogen or alkyl.
 14. A compound of claim 12,wherein the linker moiety is —C(O)—CH₂—N(H).
 15. A compound of claim 13,wherein the linker moiety is —C(O)—CH₂—N(H).
 16. A compound according toclaim 1, wherein said compound is a monochloride salt.
 17. A compoundaccording to claim 2, wherein said compound is a monochloride salt. 18.A compound according to claim 1, wherein said compound is a dichloridesalt.
 19. A compound according to claim 2, wherein said compound is adichloride salt.
 20. A method of diagnosing Alzheimer's disease in ahuman patient which comprises administering to a patient in need of suchdiagnosis a compound according to claim 1 and measuring theradioactivity arising from the administration of the compound to saidpatient either by using a gamma camera or by positron emissiontomography (PET).
 21. A process for production of a compound of formula(I) according to claim 1 comprising: reacting a compound of formula (f):

wherein R¹, R², R³, and X¹ are as defined in claim 1, with a compound offormula (b):

in the presence of a reducing agent.